Obesity has become widespread with increases in prevalence across all developed nations (Bouchard, C (2000) N Engl J Med. 343, 1888-9). According to the Center for Disease Control (CDC), over 60% of the United States population is overweight, and greater than 30% are obese. For affected persons, the problem often begins in childhood, and continues for life. Major contributors are believed to be increased consumption of high calorie foods and a more sedentary life style. However, neither of these alone or together are sufficient to explain the rise in obesity and subsequent or concomitant obesity-related disorders, such as, e.g., type II diabetes mellitus, metabolic syndrome, hypertension, cardiac pathology, and non-alcoholic fatty liver disease. According to the National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK) approximately 280,000 deaths annually are directly related to obesity. The NIDDK further estimated that the direct cost of healthcare in the U.S. associated with obesity is $51 billion. In addition, Americans spend $33 billion per year on weight loss products. The prevalence of obesity continues to rise at alarming rates.
It is estimated that between 20-25% of American adults (about 47 million) have metabolic syndrome, a complex condition associated with an increased risk of vascular disease. Metabolic syndrome is also known as Syndrome X, metabolic syndrome X, insulin resistance syndrome, or Reaven's syndrome. Metabolic syndrome is generally believed to be a combination of disorders that affect a large number of people in a clustered fashion. The symptoms and features of the syndrome include at least three of the following conditions: diabetes mellitus type II; impaired glucose tolerance or insulin resistance; high blood pressure; central obesity and difficulty losing weight; high cholesterol; combined hyperlipidemia; including elevated LDL; decreased HDL; elevated triglycerides; and fatty liver (especially in concurrent obesity). Insulin resistance is typical of metabolic syndrome and leads to several of its features, including glucose intolerance, dyslipidemia, and hypertension. Obesity is commonly associated with the syndrome as is increased abdominal girth, highlighting the fact that abnormal lipid metabolism likely contributes to the underlying pathophysiology of metabolic syndrome.
Metabolic syndrome was codified in the United States with the publication of the National Cholesterol Education Program Adult Treatment Panel III (ATP III) guidelines in 2001. On a physiologic basis, insulin resistance appears to be responsible for the syndrome. However, insulin resistance can be defined in a myriad of different ways, including impaired glucose metabolism (reduced clearance of glucose and/or the failure to suppress glucose production), the inability to suppress lipolysis in tissues, defective protein synthesis, altered cell differentiation, aberrant nitric oxide synthesis affecting regional blood flow, as well as abnormal cell cycle control and proliferation, all of which have been implicated in the cardiovascular disease associated with metabolic syndrome. At least at present, there is no obvious molecular mechanism causing the syndrome, probably because the condition represents a failure of one or more of the many compensatory mechanisms that are activated in response to energy excess and the accumulation of fat.
Individuals at risk for metabolic syndrome include those who exhibit central obesity with increased abdominal girth (due to excess visceral adiposity) of about more than 35 inches in women and more than 40 inches in men. Individuals at risk for metabolic syndrome also include those that have a BMI greater than or equal to 30 kg/M2 and may also have abnormal levels of nonfasting glucose, lipids, and blood pressure.
Although certain bacterial associations have been examined for these and related conditions, the role of bacterial microbiota in these conditions has not been clearly understood or appreciated. Thus, there remains a need for methods for diagnosing, treating and preventing conditions such as obesity, metabolic syndrome, insulin-deficiency or insulin-resistance related disorders, glucose intolerance, diabetes mellitus, non-alcoholic fatty liver, abnormal lipid metabolism, atherosclerosis, and related disorders.
The average human body, consisting of about 1013 cells, has about ten times that number of microorganisms. The ˜1014 microbes that live in and on each of our bodies belong to all three domains of life on earth—bacteria, archaea and eukarya. The major sites for our indigenous microbiota are the intestinal tract, skin and mucosal surfaces such as nasal mucosa and vagina as well as the oropharynx. By far, the largest bacterial populations are in the colon. Bacteria make up most of the flora in the colon and 60% of the dry mass of feces. Probably more than 1000 different species live in the gut. However, it is probable that >90% of the bacteria come from less than 50 species. Fungi and protozoa also make up a part of the gut flora, but little is known about their activities. While the microbiota is highly extensive, it is barely characterized. Consequently, the Roadmap of the National Institutes of Health (NIH) includes the “Human Microbiome Project” to better characterize our microbial communities and the genes that they harbor (our microbiome) and better understand its relation to both human health and disease. Reviewed in Dethlefsen et al., Nature, 2007, 449:811-818; Turnbaugh et al., Nature, 2007, 449:804-810; Ley et al., Cell, 2006, 124:837-848.
Studies show that the relationship between gut flora and humans is not merely commensal (a non-harmful coexistence), but rather often is a mutualistic, symbiotic relationship. Although animals can survive with no gut flora, the microorganisms perform a host of useful functions, such as training the immune system, preventing growth of harmful species, regulating the development of the gut, fermenting unused energy substrates, metabolism of glycans and amino acids, synthesis of vitamins (such as biotin and vitamin K) and isoprenoids, biotransformation of xenobiotics, and producing hormones to direct the host to store fats. See, e.g., Gill et al., Science. 2006, 312:1355-1359; Zaneveld et al., Curr. Opin. Chem. Biol., 2008, 12(1):109-114; Guarner, Digestion, 2006, 73:5-12; Li et al., Proc. Natl. Acad. Sci. USA, 2008, 105:2117-2122; Hooper, Trends Microbiol., 2004, 12:129-134; Mazmanian et al., Cell, 2005, 122:107-118; Rakoff-Nahoum et al., Cell, 2004, 118:229-241. It is therefore believed that changes in the composition of the gut microbiota could have important health effects (Dethlefsen et al., PLoS Biology, 2008, 6(11):2383-2400). Indeed, a correlation between obesity and changes in gut microbiota has been observed (Ley et al., Proc Natl Acad Sci USA, 2005; 102:11070-11075; Bäckhed et al., Proc Natl Acad Sci USA, 2004; 101:15718-15723). Furthermore, in certain conditions, some microbial species are thought to be capable of directly causing disease by causing infection or increasing cancer risk for the host (O'Keefe et al., J Nutr. 2007; 137:175S-182S; McGarr et al., J Clin Gastroenterol., 2005; 39:98-109).
A substantial number of species in vertebrate microbiota is very hard to culture and analyze via traditional cultivation-based studies (Turnbaugh et al., Nature, 2007, 449:804-810; Eckburg et al., Science, 2005, 308:1635-1638). In contrast, broad-range PCR primers targeted to highly conserved regions makes possible the amplification of small subunit rRNA gene (16S rDNA) sequences from all bacterial species (Zoetendal et al., (2006) Mol Microbiol 59, 1639-1650), and the extensive and rapidly growing 16S rDNA database facilitates identification of sequences to the species or genus level (Schloss and Handelsman, (2004) Microbiol Mol Biol Rev 68, 686-691). Such techniques can also be used for identifying bacterial species in complex environmental niches (Smit et al., (2001) Appl Environ Microbiol 67, 2284-2291), including the human mouth, esophagus, stomach, intestine, feces, skin, and vagina, and for clinical diagnosis (Harris and Hartley, (2003) J Med Microbial 52, 685-691; Saglani et al., (2005) Arch Dis Child 90, 70-73).
Much of the microbiota is conserved from human to human, at least at the level of phylum and genus (for a general description of human microbiota see, e.g., Turnbaugh et al., Nature 2007; 449:804-810; Ley et al., Nature 2006; 444:1022-1023; Gao et al., Proc Natl Acad Sci USA 2007; 104:2927-32; Pei et al., Proc Natl Acad Sci USA 2004; 101:4250-4255; Eckburg et al., Science 2005; 308:1635-1638; Bik et al., Proc Natl Acad Sci USA 2006; 103:732-737). A major source of the human microbiota is from one's mother (for a summary of typical maternal colonization patterns see, e.g., Palmer et al., Plos Biology 2007; 5:e177; Raymond et al., Emerg Infect Dis 2004; 10:1816-21), and to a lesser extent from one's father and siblings (for examples of typical colonization patterns see, e.g., Raymond et al., Emerg Infect Dis 2004; 10:1816-21; Raymond et al., Plos One 2008; 3:e2259; Goodman et al., Am J Epidemiol 1996; 144:290-299; Goodman et al., Lancet 2000; 355:358-362). However, many of the natural mechanisms for the transmission of these indigenous organisms across generations and between family members have diminished with socioeconomic development. The impediments include: childbirth by caesarian section, reduced breast-feeding, smaller family size (fewer siblings), reduced household crowding with shared beds, utensils, in-door plumbing.
The vertebrate intestinal tract has a rich component of cells involved in immune responses. The nature of the microbiota colonizing experimental animals or humans affects the immune responses of the populations of reactive host cells (see, e.g., Ando et al., Infection and Immunity 1998; 66:4742-4747; Goll et al., Helicobacter. 2007; 12:185-92; Lundgren et al., Infect Immun. 2005; 73:523-531).
The vertebrate intestinal tract also is a locus in which hormones are produced. In mammals, many of these hormones related to energy homeostasis (including insulin, glucagon, leptin, and ghrelin) are produced by organs of the intestinal tract (see, e.g., Mix et al., Gut 2000; 47:481-6; Kojima et al., Nature 1999; 402:656-60; Shak et al., Obesity Surgery 2008; 18(9):1089-96; Roper et al., Journal of Clinical Endocrinology & Metabolism 2008; 93:2350-7; Francois et al., Gut 2008; 57:16-24; Cummings and Overduin, J Clin Invest 2007; 117:13-23; Bado et al., Nature 1998; 394:790-793).
Changing of the microbiota of the intestinal tract appears to affect the levels of some of these hormones (see, e.g., Breidert et al., Scand J Gastroenterol 1999; 34:954-61; Liew et al., Obes. Surg. 2006; 16:612-9; Nwokolo et al., Gut. 2003; 52, 637-640; Kinkhabwala et al., Gastroenterology 132:A208). The hormones affect immune responses (see, e.g., Matarese et al., J Immunol 2005; 174:3137-3142; Matsuda et al., J. Allergy Clin. Immunol. 2007; 119, S174) and adiposity (see, e.g., Tschop et al., Nature 2000; 407:908-13).
Hydroxypropylmethylcellulose (HPMC) is modified cellulose fiber that produces viscous solutions in the gastrointestinal tract. It has been demonstrated that high viscosity (HV) HPMC consumed as part of a meal reduced peak blood glucose concentrations in subjects with type 2 diabetes compared with a cellulose control (Reppas et al., Diabetes Res. Clin. Pract., 1993, 22:61-9). It has been further demonstrated that HPMC reduced weight gain and insulin resistance in diet-induced obese mice and syrian hamsters fed a high fat (HF) diet similar in fat content to the American diet. (Hung et al., J Diab 2009; 1(3):194-206); Kim et al., FASEB J., 2009, Meeting Abstracts, Abstract 212.2).
PCT Pat. Appl. Publ. Nos. WO 2008/051793 and WO 2008/051794 disclose the use of HPMC and other water-soluble and water-insoluble cellulose derivatives for preventing or treating metabolic syndrome and related conditions. See also U.S. Pat. Nos. 5,576,306; 5,585,366; 6,899,892; 5,721,221. PCT Pat. Appl. Publ. No. WO 2004/022074 discloses the use of a composition comprising a non-glucose carbohydrate and soluble fiber or a mixture of pectin and soluble fiber for controlling metabolic syndrome, diabetes mellitus and obesity, and for the promotion of weight loss or maintenance of the desired body weight.
Obesity rates have been increasing in the United States (Ogden et al., JAMA 2014; 311:806), and recent studies have shown that the intestinal microbiota can increase fat mass (Turnbaugh et al., Nature 2006; 444:1027-1131) either through increased energy extraction or altered metabolic and inflammatory signaling (Cox et al., Cell Metab 2013; 17:883-894), suggesting that altered microbiota could be contributing to the obesity epidemic. In addition, farmers have been using low dose antibiotics for decades to increase weight gain in livestock, further indicating that alterations in the microbiota drive weight gain. Importantly, these effects are mediated by the microbiota, not antibiotics per se, since low dose antibiotics does not increase weight in germ-free animals (Coates et al., Br J Nutr 1963; 17:141-150).
The early-life microbiota plays a crucial developmental role in shaping metabolism, but the mechanism of action has yet to be fully understood. Mammalian species have co-evolved with their gut microbiota (Ley et al., Science 2008; 320:1647-1651) and much of the founding microbial population is transferred vertically from mother to child (Pantoja-Feliciano et al., ISME J 20132; 7:1112-1115). Disruptions to the early-life microbiota, such as from antibiotics or delivery by Cesarean section, significantly increase the risk of being overweight later in childhood in the human population (see, e.g., Azad et al., Int J Obes 2014; 1-9; Bailey et al., JAMA Pediatrics 2014, doi:10.1001/jamapediatrics.2014.1539; Ajslev et al., Int J Obes 2011; 35:522-529; Blustein et al., Int J Obes 2013; 37:900-906; Trasande et al., Int J Obes 2013; 37:16-23; Cox et al., Nat Rev Endocrinol 2015, 11:182-190). Antibiotic use is high in the United States, especially in infancy, with the average child receiving three courses of antibiotics by the age of two (Hicks et al., New Engl J Med 2013; 368:1461-1462), highlighting the need to understand specific microbial components that can program towards or protect from obesity.
The intestinal microbiota plays a role in shaping metabolism and immunity throughout life, and is recognized as a novel therapeutic target to stem the rising obesity epidemic, to boost immune responses, or to combat allergic and autoimmune diseases. However, microbiota-based therapies are limited to a narrow selection of bacteria and fungi. Because of the specificity of the microbiota-host interactions, it is imperative to obtain beneficial organisms in pure culture in order to consistently deliver them as therapeutics. Compared to the vast number of organisms in the GI tract, there are relatively few genera administered within probiotics available on the market today.